The Study Of Pancreatic Stellate Cell In The Mechanism Of Pancreatic Fibrosis And Intervention Of Salvianolic Acid B

Stellate cells derive their name from their shape and are also present in several organs,including the liver,kidney,pancreas and lung.Cells in the pancreas that were similar to hepatic stellate cells(HSCs) in that they were fat-storing cells were first observed with the use of autofluorescence and electron microscopy in 1982. Subsequently,two landmark reports described the isolation and initial characterization of what have henceforth been termed pancreatic stellate cells(PSCs).PSCs express intermediate filament proteins that usually characterize several cell types-for example, desmin,which characterizes myocytes;GFAP,which characterizes astrocytes;vimentin, which characterizes cells such as leukocytes,fibroblasts,and endothelial cells;and nestin,which characterizes neuroepithelial stem cells.The expression of such a diversity of intermediate filament proteins highlights that PSCs have a broad range of potential properties,including contractility,the presence of cellular extensions to sense their environment,the potential to elaborate ECM components,and the potential to proliferate.However,it is important to note that these markers have clear limitations and that there are species differences.Activation of quiescent PSCs,which occurs when primary PSCs are cultured and in the pancreas as a consequence of pancreatic injury,is associated with several morphologic changes,including nuclear enlargement and enhanced prominence of the endoplasmic reticulum network.Furthermore,in situ hybridization and immunohistochemical studies indicated that activated PSCs express alpha-SMA(also known as ACTA2) and collagen typeâ… ,therefore marking these cells as a source of fibrosis in chronic pancreatitis and pancreatic adenocarcinoma.Chronic pancreatitis is a progressive chronic inflammatory disease of the pancreas characterized by glandular atrophy,ductal changes,and extensive fibrosis.Although the clinical,morphological,and etiological characteristics of chronic pancreatitis are well known,the pathogenic mechanism has remained elusive.Pancreatic fibrosis is regulated by a balance between production and degradation of the ECM.ECM secreted by PSCs is degraded by matrix metalloproteinases(MMPs),and their activity is blocked by tissue inhibitors of matrix metalloproteinases(TIMPs).Multiple studies have identified several major signaling pathways involved in the regulation of PSC function. Mitogen-activated protein kinase(MAPKs) are key mediators of activating signals initiated by growth factors,angiotensinâ…¡,and ethamol.Other signaling pathways regulating PSC activation include PI3K,RHO kinase,the JAK/STAT pathways,the activator protein-1 and NF-κB pathways,and the TGF-β/SMAD-related pathways.Salvia miltiorrhiza(also known as Danshen),one of the well-known Chinese herbal medicines,is officially listed in the Chinese Pharmacopoeia and used as an important component of certain recipes for treatment of cardiovascular disorders as well as inflammatory diseases such as arthritis,and chronic hepatitis.Salvianolic acid B(Sal-B), one of water soluble component from Danshen,its molecular formula C36H30O16 with MW718,is the major and most active radical scavenging and antioxidant from Danshen. Previous studies have showed that Sal-B can effectively reverse liver fibrosis induced by tetrachloride carbon or di-methylnitrosamine,ameliorate oxidative damage, eliminate ROS accumulation in hepatocytes,and attenuate hepatic stellate cells activation,potentially conferring hepatoprotective and anti-fibrogenic effects.As we all known,there is no effective therapy for chronic pancreatitis,and little progress has recently been made in the field of diagnosis and therapy of chronic pancreatitis.On the basis of the above-mentioned evidence,we reasoned that Sal-B might prevent or mitigate chronic pancreatitis.Materials and Methods:1) Induction of chronic pancreatitis and activation of pancreatic stellate cell in modelChronic pancreatitis was induced using the method described Puig-Divi et al. Animals were sacrificed at the end of two,four and eight weeks.Pancreas were quickly removed,portions immediately snap-frozen and stored at -80℃until the analyses were carried out.Collagen typeâ… mRNA was evaluated by reverse transcriptase-polymerase chain reaction(RT-PCR).Other parts were fixed in 10%formalin and used for light microscopic using a previously described scoring system:(0=absent,1=a lesion slightly shown in the 2-3 lobules,2=a lesion widely shown in less than one haif lobules,and 3=a lesion shown in more than one half lobules or with destruction of lobular architecture;overall results are expressed as the total scores of rats assigned to each of the three histologic grades).And the activation of PSCs was evaluated by immunohistochemical technique.The rats received 0.4 ml solvent(phosphate-buffered saline,PBS,pH 8.0 with 10%ethanol) were used as controls.2) Treatment of salvianolic acid B in modelChronic pancreatitis was induced using the same method.Thirty rats in the experimental chronic pancreatitis groups(including group 1 and group 2),12 rats in the solvent treated group and 12 rats in the sham-operation group were used in order to reach 10 rats in each group.The rats in the group 3 received 0.4 ml solvent,and the rats in the group 4 were sham-operation without TNBS or solvent.Each group included 10 animals.Rats that died within four weeks after TNBS(group 1 and group 2) or solvent (group 3) administration were replaced with new ones to maintain 10 animals in each group until the end of four weeks.Mortality rate of the experimental groups(including group 1 and group 2) was 13%(4/30),and there were no death in other groups.Because chronic pancreatitis develops two to three weeks after TNBS administration,treatment was started at the beginning of five weeks.The dose of Sal-B was daily 10mg/kg body weight for rats(i.e.10 times adult clinic therapeutic dose).At the beginning of five weeks,the rats in the group 2 was treated with about 2 ml of Sal-B by garage,and the rats in other groups received the same volume of water for eight weeks.One rat from the group 1,which died between four and twelve weeks,was excluded from the study.Body weight was monitored weekly.Animals were sacrificed at the end of twelve weeks.Serum was kept at -20℃until the assays were carried out using an automatic biochemical analyzer according to the instructions of the manual(TBA-40FR,Tokyo,Japan).Pancreas were quickly removed, freed from fat and lymph nodes and weighted.Portions immediately snap-frozen and stored at -80℃until the analyses were carried out.Collagen typeâ… mRNA was evaluated by reverse transcriptase-polymerase chain reaction(RT-PCR).Other parts were fixed in 10%formalin and used for light microscopic using the same scoring system.And the activation of PSCs was evaluated by immunohistochemical technique.3) Cell proliferation assayCell growth experiments were performed using the 3-(4,5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay.LTC-14(A present from Professor Robert Jaster) were counted and seeded at a density of 3800 cells/well onto 96-well plates containing Iscoves modified Dulbeccos medium(IMDM) supplement with 10%fetal bovine serum,non-essential amino acid,and cultured at 37℃in a humidified environment containing 5%CO2.After treatment with various concentrations of salvianolic acid B for 48 hours,twenty microliter of MTT(5mg/ml) was added to each well for an additional 4 hours.Formazan products were solubilized with dimethyl sulphoxide(DMSO),and the optical density was measured at 570 nm.4) Treatment of salvianolic acid B in PSCLTC-14 cells were counted and seeded at a density of 4×10⁴ cells/well onto 6-well plates containing IMDM supplement with 10%fetal bovine serum,non-essential amino acid,and cultured at 37℃in a humidified environment containing 5%CO₂.After treatment with three concentrations(1μmol/L,10μmol/L,100μmol/L) of salvianolic acid B for 48 hours,alpha smooth muscle actin and collagen typeâ… mRNA was evaluated by RT-PCR,and extracellular signal-regulated kinase-1/2(ERK1/2) was evaluated using western blot technique.Results:1) Induction of chronic pancreatitis and activation of pancreatic stellate cell in modelIn pancreatic tissue after two weeks after surgical procedure,there were inflammatory cell infiltration,few alpha-smooth muscle actin positive cells except for vascular smooth muscle cells and higher level of collagen typeâ… mRNA compared with the control,but without atrophy and fibrosis.In pancreatic tissue after four weeks after surgical procedure,there were inflammatory cell infiltration,prominent fibrosis and atrophy,many alpha-smooth muscle actin positive cells and higher level of collagen typeâ… mRNA compared with the control.In pancreatic tissue after eight weeks after surgical procedure,the changes were similar with that of four weeks,but more obvious.2) Treatment of salvianolic acid B in modelHistopathologic analysesAt the end of twelve weeks,segmental glandular atrophy was prominent in the group 1.Interstitial edema,mononuclear inflammatory cell infiltration,destruction of acini,and intralobular or interlobular and periductal fibrosis were also observed.Sal-B clearly improved pancreatic histological findings in experimental pancreatitis rats,as demonstrated by light microscopy inspection.The animals in the group 3 and the group 4 had significantly less pancreatic injury and fibrosis when compared with other groups. The histopathologic scores were higher in the TNBS treated group than in the TNBS+ Sal-B treated group(p＜0.01).Subgroup analysis of pathological scores revealed that the rats in the TNBS+Sal-B treated group had significantly lower scores than the rats in the TNBS treated group(p＜0.05),except inflammation(p＞0.05).Activation of pancreatic stellate cellsImmunostaining for alpha-SMA was performed to identify the activated PSCs.In the TNBS treated group,it revealed a marked proliferation of alpha-SMA positive cells in the pancreas.However,there were few alpha-SMA positive cells in the TNBS+ Sal-B treated group at the end of twelve weeks except for vascular smooth muscle cells. There were also few alpha-SMA positive cells in the pancreas of group 3 and group 4 rats.Expression of collagen typeâ… mRNASal-B suppressed the expression of collagen typeâ… mRNA in pancreatic tissue compared with group 1(p＜0.01).Compared with group 3 and group 4,pancreatic collagen typeâ… mRNA obviously increased in group 1(p＜0.01),but not in group 2 (p＞0.05).3) Proliferation of pancreatic stellate cellsAccording to MTT assay,1μmol/L Sal-B had no effect on the proliferation of PSCs (p＞0.05).10μmol/L and 100μmol/L Sal-B significantly suppressed PSCs proliferation compared with the control(p＜0.01).4) Treatment of salvianolic acid B in PSCExpression of alpha-SMA and collagen typeâ… mRNASal-B suppressed expression of collagen typeâ… and alpha smooth muscle actin in pancreatic stellate cells compared with control(p＜0.01).Expression of alpha-SMA and collagen typeâ… mRNA decreased gradually with increasing the concentration of Sal-B. Suppression of ERK 1/2 activity Sal-B suppressed activity of ERK1/2 in pancreatic stellate cells,and decreased collagen typeâ… protein expression compared with control(p＜0.01).The activity of ERK1/2 decreased with increasing increasing the concentration of Sal-B.Conclusion:1) Chronic pancreatitis model induced by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts had the similar character of human chronic pancreatitis. Pancreatic stellate cell had an important role in chronic pancreatitis.2) Salvianolic acid B suppressed the expression of alpha-smooth muscle actin and the activation of pancreatic stellate cell by decreasing the activity of extracellular signal-regulated kinase 1/2.3) Salvianolic acid B decreased collagen typeâ… and attenuated the morphological pancreatic damage by suppressing the activation of pancreatic stellate cells.